Glucan is a generic term for polysaccharides in which the saccharide components consist only of D-glucose. Examples of representative glucans include starch, cellulose, and the like. Starch is an α-glucan in which saccharide components are linked by α-glucoside bonds. In starch, amylose, which is a straight-chain α-1,4-glucan, and amylopectin, having a branched structure, are present. The abundance ratio of amylose to amylopectin varies depending on the plant storing the starch. Therefore, it is very difficult to obtain starch containing amylose and amylopectin at an arbitrary composition ratio. If amylose can be stably produced, such amylose can be mixed with commercially available starch to produce starch having an arbitrary amylose content.
Conventionally, amylose and amylopectin having an arbitrary structure are produced by utilizing the action of hydrolyzing enzymes, transferases, and the like. However, reports on structural alteration of starch by extending its α-1,4-glucan chain are limited. It is useful to develop a method for extending an α-1,4-glucan chain efficiently since not only can amylose be produced but also the structure of starch can be arbitrarily modified.
It is known that in starch-containing foods, the content and structure of amylose in the starch has a great influence on the physical properties of the food. However, the content and structure of amylose in the starch is determined by the starch utilized as a raw material. If the content and structure of amylose can be arbitrarily changed, development of foods having novel mouthfeel can be expected.
Insoluble amylose is expected to have the same function as that of dietary fiber, and can also be expected to be utilized in health foods. Further, amylose has a feature that the amylose can include, for example, iodine, fatty acid, or the like in the molecule. Therefore, amylose is expected to be used in the fields of medicaments, cosmetics, and sanitary products. Amylose can also be utilized as a raw material for the production of cyclodextrin and cycloamylose having the same inclusion capacity as that of amylose. Further, films containing amylose have tensile strength not less than that of general-purpose plastics, and amylose is a very promising material for biodegradable plastics. Thus, amylose is expected to have a number of applications. However, it is difficult to obtain substantially pure amylose and such amylose is very expensive. Therefore, such amylose is only distributed as reagent grade, and is hardly utilized as industrial material. Accordingly, there is a demand for a method for producing amylose in a stable and inexpensive manner.
There are some known methods for producing amylose. Amylose is present in starch in a proportion of about 0 to 70%. Amylose can be extracted from natural material starch using precipitating agents, such as butanol, by a method described in T. J. Schoch et al., J. American Chemical Society, 64, 2957 (1942). However, this extraction operation is complicated and has a low yield. Further, it is difficult to obtain straight-chain glucan containing no α-1,6-glucoside bonds by the extraction operation. Furthermore, it is difficult to extract straight-chain glucan having a narrow molecular weight distribution.
As a method for extending an α-1,4-glucan chain enzymatically, there is a synthetic method in which a sugar nucleotide is used as a substrate, and the sugar moiety is transferred to maltotetraose or the like as a primer by means of glycogen synthase, starch synthase, or the like. However, this method has a disadvantage that sugar nucleotides, which are used as a substrate, are very expensive and therefore cannot be industrially utilized.
There is a method for synthesizing an α-1,4-glucan chain by transferring the glycosyl group of α-glucose-1-phosphate to a primer, such as maltoheptaose or the like by means of glucan phosphorylase (GP) derived from potato.
Further, a method in which glucose-1-fluoride is used as a substrate and sucrose phosphorylase (SP) and glucan phosphorylase are simultaneously allowed to act on a primer, is disclosed (U.S. Pat. No. 5,405,449 and EP 0590736).
These synthesis methods have an advantage that the ratio of a substrate to a primer in a reaction solution at the start of reaction is arbitrarily set so that the molecular weight of the resultant straight-chain glucan can be controlled. However, the substrates, α-glucose-1-phosphate and glucose-1-fluoride, are expensive, and therefore are not suitable for inexpensive production of a straight-chain α-1,4-glucan which may be utilized in a wide range of industries.
As a method for producing straight-chain glucan in a more inexpensive manner, a method in which sucrose phosphorylase and glucan phosphorylase are simultaneously allowed to act on a primer and sucrose (hereinafter referred to as the SP-GP method) has been disclosed (Waldmann, H. et al., Carbohydrate Research, 157 (1986) c4-c7). Waldmann et al. synthesized straight-chain glucan from sucrose at a high yield using sucrose phosphorylase derived from the genus Leuconostoc (Leuconostoc mesenteroides) and glucan phosphorylase derived from potato tuber. The SP-GP method of Waldmann et al. is promising in that an inexpensive substrate can be used to produce a straight-chain glucan, but has some problems which require improvement as shown below.
First of all, since a large amount of enzyme is used, the production cost is high and inexpensive production is not possible. To solve this problem, the amount of an enzyme used needs to be reduced or the amylose productivity per unit enzyme needs to be improved by studying the reaction conditions.
One of factors which determine the amount of enzyme used or the amylose productivity per enzyme in the SP-GP method is, for example, the concentration ratio of inorganic phosphate to sucrose, which is a substrate for SP, in a solution at the start of reaction. In the prior art of Waldmann et al., inorganic phosphate having a considerably low concentration compared to the sucrose concentration in a solution at the start of reaction is used, so that glucan is produced at a high yield. In the SP-GP method in which sucrose is first converted to glucose-1-phosphate and then the glucose-1-phosphate is converted to glucan, if high-concentration inorganic phosphate is used, an intermediate, glucose-1-phosphate, accumulates at a high concentration. Therefore, it is considered that the yield of the final product, glucan, is reduced. Therefore, it is considered that a low inorganic phosphate concentration should be adopted in the conventional SP-GP method. Before the present invention, there was no disclosure of what influence a change in the concentration ratio of sucrose to inorganic phosphate in a solution at the start of reaction has on the amount of an enzyme used or the amylose productivity per enzyme, much less prediction of the effect of such an change.
A method in which two types of phosphorylase are combined similarly to the SP-GP method and carbohydrate is synthesized via inorganic phosphate has been reported. For example, Chaen et al. (Journal of Bioscience and Bioengineering, 92 (2001) 177-182) discloses a method for synthesizing kojioligosaccharide from maltose using a combination of maltose phosphorylase and kojibiose phosphorylase. Chaen et al. describes that the lower the inorganic phosphate concentration in a solution at the start of reaction, the higher the yield of the reaction product, i.e., kojioligosaccharide. Thus, it is conventionally considered that in a reaction system in which two phosphorylases are combined, the inorganic phosphate concentration in a solution at the start of reaction is preferably low in order to increase the yield of the final product.
A second factor which determines the amount of enzyme used or the amylose productivity per enzyme in the SP-GP method is reaction temperature. Generally, in enzyme reactions, the higher the reaction temperature, the greater the reaction rate. Therefore, it is desirable that reactions are conducted under high temperature conditions. However, since enzyme proteins are unstable to heating, an actual enzyme reaction is conducted in a temperature range in which the enzyme proteins are not thermally inactivated. In the prior art of Waldmann et al., sucrose phosphorylase derived from the genus Leuconostoc was used, and a glucan synthesis reaction was conducted at 37° C. by taking into account the thermal stability of this enzyme. Before the present invention, it had not been disclosed what influence a change in the sucrose concentration of the reaction solution has on the stability of sucrose phosphorylase, and the effect thereof could not be predicted. Further, there had been no disclosure on the thermal stability of sucrose phosphorylase derived from the genus Streptococcus. Therefore, it was not possible to predict any effect of utilizing this sucrose phosphorylase in glucan synthesis.
As a second problem, there is an operability problem. Glucan, particularly amylose, ages to become insoluble, resulting in precipitation or formation of a gel. It is well known that the aging rate is dependent on temperature. When reaction temperature is low, an operability problem arises in subsequent stages after the production, such as gelation of amylose solution. Therefore, the reaction temperature is preferably as high as possible. However, in the prior art of Waldmann et al., the reaction temperature at which amylose is produced is problematically as low as 37° C.
Out of glucans, when a straight-chain amylose without a branched structure is to be specifically produced, straight-chain malto-oligosaccharide has to be used as a primer. In the prior art of Waldmann et al., purified maltoheptaose is utilized as straight-chain malto-oligosaccharide. However, purified maltoheptaoseis only distributed as reagent grade and is very expensive. Inexpensive primer candidates include a mixture of malto-oligosaccharide obtained by hydrolyzing starch appropriately. However, it is known that for a number of glucan phosphorylases, only malto-oligosaccharides having a degree of polymerization greater than or equal to that of maltotetraose can be utilized as a primer, but malto-oligosaccharides having a degree of polymerization smaller than or equal to that of maltotriose cannot be utilized as a primer. The malto-oligosaccharide mixture contains maltotriose, maltose and glucose which do not function as a primer in addition to malto-oligosaccharide having a degree of polymerization greater than or equal to that of maltohexaose which can function as a primer. Further, it is known that glucose contained in the malto-oligosaccharide mixture is an inhibitor of sucrose phosphorylase. Thus, whether or not the malto-oligosaccharide mixture containing maltotriose, maltose and glucose incapable of functioning as a primer and glucose which is an inhibitor of sucrose phosphorylase, can function effectively in the SP-GP method had not been disclosed before the present invention, and the effectiveness thereof could not be easily speculated.
In the case of a method of the present invention, a large amount of fructose is secondarily produced along with glucan in the reaction solution after ending an enzyme reaction. Therefore, in the case of industrial glucan production using the method of the present invention, a process for efficiently purifying glucan after a glucan synthesis reaction step is essential. In the prior art of Waldmann et al., when purifying amylose, a method for selectively precipitating amylose using butanol is utilized. However, when amylose is industrially mass produced, a method utilizing an organic solvent is not an excellent method in terms of costs, safety to a human body, and environmental issues. No method using no organic solvent has been disclosed.